In the medical field, there is high expectation for diagnosis, treatment or prevention of diseases using genes (gene diagnosis). By examining defects or changes in causal genes of particular diseases for example, the gene diagnosis allows for diagnosis, treatment or prevention before onset of disease or at early stages of disease. The gene diagnosis also realizes what is known as tailor made medicine, which is based on relationships between genotype and disease as revealed by human genome analysis. In view of this, a further development is expected for easy detection of genes and/or easy determination of genotypes.
As a tool for gene detection and/or genotype determination, a DNA chip (DNA microarray, or more generally nucleic acid immobilizing substrate) has been available that is used to hybridize a subject sample with nucleic acids immobilized on a substrate or a support. The DNA chip is considered to be useful in applications including not only basic medical science but clinical medicine, drug creation and/or preventive medicine as well.
The nucleic acid immobilizing substrate is also important for the development of nucleic acid-based nanotechnology (for example, nano wires, and nucleic acid-based nano electronics such as bio-sensors and bio-chips) (see Non-Patent Publication 1, for example).
In detection of nucleic acid based on a hybridization method, there has been proposed a method in which a sample containing a nucleic acid (target DNA) complementary to a nucleic acid probe such as a DNA fragment is immobilized on a support such as a nitrocellulose film and is allowed to react with the nucleic acid probe in a solution (see Non-Patent Publication 2, for example).
As a method by which nucleic acids used for the DNA chip are immobilized on a substrate, a method is known that directly synthesizes a nucleic acid probe on a substrate (see Non-Patent Publication 3, for example). As another method of immobilizing nucleic acids on a support, there has been known a method in which a nucleic acid probe is immobilized on a support after it has been prepared beforehand (see Non-Patent Publication 4, for example).
In another known method, a solid-phase support that has been surface-treated with a silane coupling agent including functional groups such as an amino group or an aldehyde group is covalently bonded to a nucleic acid probe having modified functional groups, after spotting with a DNA chip fabricating device (see Non-Patent Publication 5).
Specifically, the following methods have been known as the method of binding a self-organizing material (for example, nucleic acid) on a solid surface: A method in which a substrate surface is treated with silane to introduce therein a vinyl group that can bind to the nucleic acid molecule (see Non-Patent Publication 6, for example); a method in which nucleic acids are bound to a substrate using counter ions (see Non-Patent Publications 7-9, for example); a method in which pH values are chemically controlled to adjust the extent of immobilization on various types of substrate surfaces (see Non-Patent Publication 10, for example); and a method in which Al2O3 surface is treated with Na3PO4 solution to render the surface hydrophilic (see Non-Patent Publication 11, for example). As a technique for removing organic impurities from the substrate surface, there has been known a method in which the molecules on the substrate surface are modified by an oxygen plasma process which requires expensive equipment (see Patent Publications 1 and 2, for example).
In the field of electronics, there has been report that nucleic acids show strong bonding, similar to covalent bond, with respect to aluminum electrodes (see Non-Patent Publication 12, for example).
[Patent Publication 1]
WO97/38801 (published on Oct. 23, 1997)
[Patent Publication 2]
Japanese Laid-Open Patent Publication No. 2002-218976 (published on Aug. 6, 2002)
[Non-Patent Publication 1]
Storhoff, J. J. and Mirkin, C. A.: Chem. Rev. 99, 1849-1862 (1999)
[Non-Patent Publication 2]
Molecular Cloning 2nd. Ed. (Cold Spring Harbor Press)
[Non-Patent Publication 3]
Forder, S. P. A. et al, Science, 251, 767-773 (1991)
[Non-Patent Publication 4]
Schena, M. et al., Science, 270, 467-470 (1995)
[Non-Patent Publication 5]
Geo, Z. et al., Nucleic Acid Research, 22, 5456-5465 (1994)
[Non-Patent Publication 6]
Bensimon, D. et al., Physical Review Letters 74, 23, 4754-4757 (1995)
[Non-Patent Publication 7]
Ye, J. Y. et al., Analytical Biochemistry 281, 21-25 (2000)
[Non-Patent Publication 8]
Dunlap, D. D. et al., Nucl. Acid Res. 25, 3095 (1997)
[Non-Patent Publication 9]
Lyubchenko, Y. L. et al., Proc. Natl. Acad. Sci. USA 94, 496 (1997)
[Non-Patent Publication 10]
Allemand, J. F. et al., Biophysical Journal, 73, 2064-2070 (1997)
[Non-Patent Publication 11]
Yoshida, K. et al., Biophysical Journal, 74, 1654-1657 (1998)
[Non-Patent Publication 12]
Washizu, M. et al., IEEE Trans. Industr. Appl., 31, 3, 447-456 (1995)
As an application of the method described in Non-Patent Publication 3, there has been known a method in which a nucleic acid probe is synthesized on a glass slide or a silicon substrate using a photolithography technique, employed in fabrication of semiconductors, in combination with a solid-phase synthesis technique. A drawback of the on-chip synthesis method, however, is that it requires special equipment and reagents for synthesizing a nucleic acid probe on the substrate, and that it can synthesize nucleic acid probes of only about several ten bases long.
As an application of the method described in Non-Patent Publication 4, a method is known in which a nucleic acid probe that has been prepared in advance using a DNA chip fabricating device is spotted for electrostatic bonding on a surface of a solid-phase support that has been surface-treated with poly-L-lysine or the like. While such an electrostatic bonding method of nucleic acid probe allows for immobilization of long nucleic acid probes, it lacks reproducibility due to procedures of hybridization reactions.
The method described in Non-Patent Publication 5 can be used to bond the nucleic acid probe onto a solid-phase support relatively firmly. However, preparation of nucleic acid probe requires the tedious procedures of PCR amplification using oligonucleotides with modified functional groups. Further, due to difficulties of the method in preparing a nucleic acid probe extending several thousand bases, identification of long nucleic acid probes is inevitably difficult.
As described above, as the technique for binding nucleic acid molecules on a substrate surface, there have been used (1) a method of modifying molecules on a substrate surface, or (2) a method of subjecting a substrate surface with a plasma process. However, the method employing molecule modification requires many steps and large equipment. Likewise, the plasma process calls for large-scale equipment and large cost. That is, it has not been possible with the conventional techniques to directly bind the nucleic acid molecules, both easily and inexpensively, on a substrate surface.
In studying DNA structures and a complex thereof, or electrical characteristics of DNA structure, the inventors of the present invention found that it was important to immobilize or extend DNA on an atomically flat substrate in order to rule out the influence of substrate structure. Immobilization of DNA is well researched. Mica, glass, gold, and highly oriented pyrolytic graphite (HOPG) are commonly used as the substrate. Mica and HOPG can be cut very easily to provide an atomically flat substrate. However, cations (for example, magnesium ion or nickel ion) are required in order to hold molecules on the substrate made of these materials. Glass substrates also require surface modification. For example, DNA is immobilized using aminopropyl triethoxysilane (APS). By coating the glass surface, APS forms a positively charged layer with amino groups. The DNA with the phosphate groups (negatively charged) around the double helix is therefore adsorbed on the substrate by the electrostatic force. Gold particles can be used for DNA with thiol ends, because gold can form a covalent bond with the thiol group.
The present invention was made in view of the foregoing problems, and an object of the present invention is to provide a self-organizing material immobilizing substrate that can immobilize a self-organizing material on a substrate surface in a controlled manner from low density to high density, and that can be manufactured at low cost. Specifically, it is an object of the present invention to provide a method for conveniently immobilizing DNA and a method for extending DNA without chemical surface modification (for example, APS process) using other molecules, and a substrate fabricated by such methods. It is another object of the present invention to provide a method for one-dimensionally arranging fine particles on a substrate surface in a desired shape within a square micrometer.